1. Field of the Invention
The present invention relates to the field of processes and techniques connected with the application of Nuclear Magnetic Resonance (NMR), particularly for high resolution tests, and has for its object a process for the excitation and acquisition of NMR signals, particularly in light water.
2. Description of the Related Art
Multidimensional NMR has become an essential instrument for the study of molecules in solution, particularly biomolecules, and serves as a good alternative to x-ray crystallography.
Thus, it is now possible to obtain protein structures up to 30-40 kD (kilo Dalton) by using a combination of NMR tests in two dimensions, three dimensions and four dimensions (see particularly G. Marius Clore and Angela M. Gronenborn, "Progress in NMR Spectroscopy", 23, 43-1991).
A crucial step for the determination of these structures is attribution of the signals of all the protons of the molecule to be analyzed and, in particular, the protons in exchange with those of water or of the corresponding solvent.
Because these protons are exchanged with water, the study of these molecules must be effected in a solution having a proportion of at least 90% water or of corresponding solvent.
Under these conditions, the proton concentration in the water is 110 M (mols) while the concentration of the solute (molecules to be analyzed) is about 2 mM (millimols).
Important difficulties result for the observation of the signals which are about 55,000 times smaller (weaker) than the strongest signal, namely, that corresponding to the water protons or those of the solvent used.
Another disadvantageous result of this high concentration of solvent is the appearance of a phenomenon known by the name "Radiation Damping" (attenuation of radiation), which further upsets the observation of the spins that are of interest for the test.
So as to overcome these problems, the most conventional solution and the one most frequently used is the presaturation of the resonance of the water.
Nevertheless, for proteins in neutral or basic pH, this solution leads to an important attenuation of the resonances of the amide proteins and for the nucleic acids, with a total disappearance of the imine and amide protein signals.
This drawback can be avoided by using impulse sequences which do not perturb the population of the spins of the water during the relaxation times.
The different processes and methods of this type known at present can be classed in three distinct categories.
A first category of methods and processes uses selective excitation sequences such as impulsion of detection of the signal, such as particularly the process known by the name "jump and return", described by P. Plateau and M. Gueron in J. Am. Chem. Soc., 104, 7310 (1982) or also the process known under the name "11--echo" described by V. Sklenar and A. Bax in J. Magn. Reson., 74, 469 (1987).
The different processes and methods under the second category propose dephasing the transverse component of the magnetization of the water by a non-homogeneous radiofrequency field by means of locking the spins (see B. A. Masserie, G. Wider, G. Wider, G. Otting, C. Weber and K. Wuthrich, J. Magn. Reson., 85, 608-1989).
Finally, the processes and methods of the third category use technologies recently developed in the field of pulsed field gradient applications (see particularly G. W. Vuister, R. Boelens, R. Kaptein, R. E. Hurd, B. John, and P. C. M. Ziji, J. Am. Chem. Soc., 113, 9688, 1991).
Nevertheless, these three categories of known processes and methods mentioned above have numerous drawbacks in common, and also specific drawbacks.
Their principal common drawback resides in the fact that the suppression or attenuation of the solvent signal, particularly of water, is weak if a single transient is registered and that good attenuation of the signal corresponding to the solvent or to the water can be obtained only after the achievement of a complete phase cycle.
Moreover, the use of these known processes and methods recited above is limited to certain circumstances and experimental conditions.
Thus, the process "jump and return" can be practiced for NMR tests of a size or type known by the name NOESY (Nuclear Overhauser Effect Spectroscopy), but is very ineffective in association with NMR tests of the type known by the name TOCSY (Totally Correlated Spectroscopy) and ROESY (Rotating Overhauser Effect Spectroscopy) and is not in practice used for heteronuclear NMR tests.
The process using a spin locking impulse gives relatively good results for NMR tests of the types known by the names HMQC (Heteronuclear Multiple Quantum Coherence) and HSQC (Heteronuclear Single Quantum Coherence), but is difficult to use for homonuclear tests.
Finally, the processes and methods using pulsed field gradient techniques are not effective, for the suppression of the water peak, except in the framework of NMR tests of the heteronuclear type and have no utility in the framework of tests of the NOESY, TOCSY and ROESY type.
Moreover, use of these latter processes and methods gives rise to a decrease by half of the signal/noise ratio.